August 2010
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Table of Contents
Acknowledgement........................................................................................................................ 2
Disclaimer .................................................................................................................................... 2
List of abbreviations ..................................................................................................................... 6
Executive Summary ..................................................................................................................... 7
Summary from the survey ........................................................................................................7
Summary of the literature review.............................................................................................. 8
1 Background and scope of this report ...................................................................................... 10
1.1 Background ...................................................................................................................... 10
1.2 Scope of the review.......................................................................................................... 10
2 Substitution/modification of nanomaterials survey .................................................................. 12
2.1 Results of previous surveys on nanomaterials used in Australia ..................................... 12
2.2 Substitution/modification survey method .......................................................................... 14
2.3 Survey results................................................................................................................... 14
2.3.1 Work Sector ............................................................................................................... 14
2.3.2 Types of engineered nanomaterials .......................................................................... 15
2.3.3 Types of activities using nanomaterials ..................................................................... 17
2.3.4 Sources of engineered nanomaterials ....................................................................... 18
2.3.5 Health and safety considerations............................................................................... 19
2.3.6 Use of modification and substitution .......................................................................... 20
2.4 Summary from the survey ................................................................................................ 23
3 Literature review for substitution/modification options for engineered nanomaterials............. 25
3.1 Mechanisms of nanoparticle toxicity in biological systems............................................... 25
3.1.1 Importance of nanoparticle size for cell uptake ......................................................... 29
3.1.2 Importance of surface charge for cell uptake............................................................. 31
3.1.3 Importance of cell specific effects for nanoparticle uptake ........................................ 32
3.1.4 Importance of surface modification for nanoparticle uptake ...................................... 33
3.1.5 Biocompatibility and surface coatings........................................................................ 34
4 Substitution/modification options for specific engineered nanomaterials................................ 35
4.1 Carbon nanotubes............................................................................................................ 35
4.1.1 Background................................................................................................................ 35
4.1.2 Toxicology of carbon nanotubes................................................................................ 37
4.1.3 Potential substitution/modification of carbon nanotubes............................................ 38
4.1.4 Impact of modification on potential exposure levels .................................................. 42
4.1.5 Conclusions ............................................................................................................... 42
4.2 Fullerenes......................................................................................................................... 43
4.2.1 Background................................................................................................................ 43
4.2.2 Toxicology of fullerenes ............................................................................................. 44
4.2.3 Potential substitution/modification of fullerenes......................................................... 45
4.2.4 Conclusions ............................................................................................................... 45
4.3 Nano titanium dioxide (TiO2) ............................................................................................ 46
4.3.1 Background................................................................................................................ 46
4.3.2 Toxicology of nano titanium dioxide........................................................................... 46
4.3.3 Potential substitution/modification of nano titanium dioxide ...................................... 47
4.3.4 Conclusions ............................................................................................................... 48
4.4 Nano cerium dioxide (CeO2)............................................................................................. 49
4.4.1 Background................................................................................................................ 49
4.4.2 Toxicology of nano cerium dioxide ............................................................................ 49
4.4.3 Conclusions ............................................................................................................... 51
4.5 Nano zinc oxide (ZnO) ..................................................................................................... 51
4.5.1 Background................................................................................................................ 51
4
List of abbreviations
ANA
ANBF
APTES
ARCNN
BSA
CME
CNTs
CPC
CTAB
CVD
DNA
EFTEM
FITC
FR+
FTIR
Her2
HHPC
HIPCO
hMSC
HSA
IARC
IR
LDH
LDL
LMCS
NICNAS
NMs
NMR
NP
MSDS
MSN
MTT
MWCNT
OHS
PEG
PLGA
PPE
PVA
QD
RBC
RCEC
ROI
ROS
SEM
SWCNT
TEM
TPGS
TSDC
UFP
XPS
XRD
Executive Summary
In a review of the evidence on the effectiveness of workplace controls to prevent exposure
to engineered nanomaterials it was found that little focus has to date been placed on use of
substitution or modification for nanotechnology work health and safety purposes. Therefore,
Safe Work Australia commissioned RMIT to undertake a survey of the current
substitution/modification practices used in Australian nanotechnology-related activities and a
literature review in order to determine the potential substitution/modification options that may
reduce the toxicity of engineered nanomaterials used in Australia.
Summary from the survey
a) There were 38 respondents to the survey, who reported working on a range of different
types of nanomaterials. The respondents organisations were primarily universities,
commercial/industry and government research groups. The most common nanomaterials
handled are metal oxides, metals and carbon nanotubes and the most common areas of
application are into energy, medical, surface coating and textile uses.
b) Many organisations (27/35), and notably universities (20/21), manufacture their own
engineered nanomaterials, and a significant number also purchase them from overseas or
from within Australia (see Figure 3).
c) A number of respondents obtained work health and safety information about the
nanomaterials that they are using from an MSDS. The main work health and safety issues
examined for engineered nanomaterials are handling and storage, physical and chemical
properties, toxicological data and exposure controls/personal protective equipment (PPE)
(see Table D). The available information on these topics is limited.
d) Most respondents indicated that substitution/modification is used to change the functional
properties of the product (see Figure 4). A work sector analysis indicates that
substitution/modification occurs more in university research and less in commercial/industry
research which is as expected in product development.
e) The five properties that are manipulated by modifying or substituting engineered
nanomaterials by the highest number of organisations are particle size, physical properties,
agglomeration properties, chemical properties and conductive properties. A small number of
respondents indicated that they use substitution/modification to change the health or
toxicological properties (see Figure 5).
f) Adding functional groups (17 responses) and modifying surface characteristics (16
responses) are the two most popular methods for the substitution/modification of engineered
nanomaterials. Others include changing the form of the material, the particle size and
shape, and the crystalline structure (see Figure 6).
g) Australias nanotechnology activities are generally at the early stage of nanomaterial
development, i.e. more focussed on de novo research than later stages of product
development/production. However substitution/modification methodologies are well known
and used in Australia and thus there is an existing capability that might be applied more
broadly to work health and safety related purposes.
i) It is possible to modify the surface of nano silica with alkylsilylation, polymers or proteins to
increase its hydrophobic character, causing increased particle aggregation and reduced
direct membrane effects, and thereby improving its biocompatibility. Due to potential toxicity
of silica nanomaterials with high aspect ratios, consideration should also be made as to
whether nanowires may be substituted with nanospheres, while retaining functionality for a
particular application.
j) It is possible to encapsulate quantum dot cores with stable shell coatings made from
biocompatible polymers, e.g. chitosan or polyethylene glycol, to significantly reduce their
cellular uptake and degradation, and consequently their cytotoxicity, whilst retaining
functionality and useability.
Implications for work health and safety
There are known methods that can be used to substitute/modify engineered nanomaterials
that are used, or researched, in Australia. The methods of surface modification,
encapsulation, particle size control, functional group addition and crystalline phase type
control can each be employed for different engineered nanomaterials to decrease their
potential toxicity. However in some cases, such modifications may affect the functionality of
nanomaterials in relation to intended end-uses.
If the researchers, developers and manufacturers of engineered nanomaterials adopt these
methods then it is possible to re-engineer nanomaterials in the early stages of development
to reduce the potential toxicity of manufactured nanomaterials. The downstream effect of
this will be to reduce the risk posed by the use of these nanomaterials not only in the
workplace but also in the general community.
10
evaluate potential opportunities for the protection of health and safety in Australian
workplaces, and
11
12
Applications
Total volume
(Tonnes per year)
Acrylic latex
Surface coatings
10000-50000
Aluminium oxide
Printing
0.05-0.1
Aluminosilicates
Water treatment
10-50
Surface coatings
10-50
Cerium oxide
Catalysts
1-5
Iron oxide
Surface coatings
1-5
Cosmetics
<0.01
Pearl powder
Cosmetics
0.01-0.05
Phthalocyanine
Surface coatings
10-50
Polyurethane resin
Surface coatings
<0.01
Cosmetics
<0.01
Silicon dioxide
Surface coatings
10-50
Water treatment
0.05-0.1
Sodium silicates
Water treatment
0.1-0.5
Printing
1-5
Printing
0.1-0.5
Printing
0.5-1
Titanium dioxide
Water treatment
5-10
Domestic products
1-5
Cosmetics
1-5
Surface coatings
5-10
Cosmetics
1-5
Zinc oxide
13
14
University Research
Commercial/Industry Research
13
21
Government Research
15
The other engineered nanomaterials that respondents reported using are nanofibres,
metallic nanoparticles, nanomembranes, montmorillonite clays, gold nanoparticles and
nanostructured proteins.
The nanomaterials being used by the highest number of responding organisations are metal
oxides, metals and carbon nanotubes, with 71% of organisations using more than one
material.
30
No. of Responses
25
20
University Research
Commercial/Industrial Research
15
Government Research
10
5
O
th
er
M
Ca
et
al
rb
ox
on
id
N
es
Ca
an
ot
rb
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on
M
es
et
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al
an
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-w
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Na
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no
ar
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ys
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s
Na
no
w
ir e
s
Fu
l
le
G
re
ra
ne
ph
s
en
e
sh
ee
ts
16
Type of material
(from Q3)
(from Q2)
Metal Oxides
Metals
Carbonaceous
Quantum dots
Other
Titanium dioxide
Zinc oxide
Iron oxide
Cerium oxide
Alumina, porous silica
Amorphous alumina
Tin dioxide
Zirconium oxide
Nano gold
Nano silver
Iron
Cobalt
Copper
Metallic nano arrays
Nickel
Palladium
Platinum
Titanium
Zinc
CNT composites
Silk
Wool
Cellulose
Polystyrene/latex
Carbon
Nanoflex
Dendrimers for transdermal delivery
CdSe
CdS
ZnSe
CdZn
Zinc glycerite
Nanoflex
Materials for defence applications
Number of
organisations
working with the
engineered
nanomaterials
9
7
5
2
1
1
1
1
6
4
3
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
17
Number of respondents
involved in this activity.
Solar cells
6
Medical
5
Composites
5
Better metallic properties
4
Textiles
4
Plastic modification
3
Sensors
2
1 response for each of the following activities
Adsorption of waste
Batteries
Catalysis
CVD growth
Dispersion of CNTs
Drug delivery
Electric conductive devices
Electro optical studies
Electrochemical processes
Electronics
Energy
Environmental
Fluorescence
Functional devices
Functionalization
Hydrogen storage
Ionic liquids
Journalism
Nano scaffolds
Opto-electronic devices
Paints
Personal care products
Pharmaceuticals
Pro-drugs using dendrimers
Separation membranes
Size characterisation
Surface coatings
Thermal processes
Toxicity testing
UV-shielding
Water treatment
18
work health and safety reasons during manufacturing, particularly in universities. There may
also be opportunities during handling and use of the nanomaterials.
30
25
No. of Responses
20
University Research
Commercial/Industrial Research
15
Government Research
10
0
Manufacture them
From Overseas
From Australia
19
Purchasing (Q6)
Manufacturing (Q7)
20
16
14
No of Responses
12
10
No
Yes
0
Government Research
Commercial/Industrial Research
University Research
14
12
No. of Responses
10
University Research
Commercial/Industrial Research
Government Research
pa
ph
rt i
ag
ys
cl
e
ic
gl
si
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lp
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21
that it exhibits the required functional properties, e.g. the particle size of nano titania is
manipulated in order to optimise its optical properties when used as a sun screening agent.
The five optional answers relating directly to work health and safety, hazardous,
toxicological or environmental properties were either the least often selected or not selected
at all.
In Question 10 respondents were asked: What approaches do you use to
modify/substitute the nanomaterials? Please tick all options that apply. Respondents were
offered a list of six choices plus other.
There were 77 responses given from 22 respondents (see Figure 6), including two
responses using the other option which specifically indicated security in confidence.
18
16
14
No of Responses
12
University Research
10
Commercial/Industrial Research
8
Government Research
6
4
2
er
th
llin
e
cr
ys
ta
pa
M
od
ify
ng
e
C
ha
ct
u
st
ru
sh
a
rti
cl
e
rti
cl
e
pa
ng
e
C
ha
re
pe
si
ze
l
ria
at
e
m
th
e
fo
ng
e
C
ha
od
ify
su
r
Ad
d
fu
fa
ce
rm
of
ch
a
nc
tio
na
ra
ct
e
lg
ro
u
ris
tic
s
ps
22
As information progressively becomes available in the scientific and public domain about
the potential use of a substitution and/or modification technique for specific engineered
nanomaterials, such techniques are more likely to also be used as a means of reducing the
hazards of nanomaterials.
Additional comments were requested in Question 11. Respondents were asked; If there is
anything you would like to add about nanomaterial modification/substitution that you would
like us to specifically consider, please write it here.....
There were five responses to this question, with respondents 1-4 working in research; these
are given in Table E.
Table E: Additional comments about the substitution/modification of engineered
nanomaterials
Respondent
1
3
4
5
Response
As a fundamental research institute, it is important that we have access to
all types of nanomaterials to determine their uses and their safety/toxicity. I
would like to see some guidelines for the researchers to protect their
health from potentially toxic nanomaterials.
Most modifications performed in our laboratory are considered standard
and in fact important avenues of research. Any dangers involved in the
modification of nanomaterials seem parallel to chemical research of any
other kind.
The nanoparticles are dispersed in molten polymer using shear to disperse
them.
In general the use, manufacture and substitution/modification of
nanomaterials is no different to that of ordinary chemistry based materials.
Yes, a lot more research into the OHS effects of working with
nanomaterials
Of the responses to this question (see Table E), answers 2 and 4 indicate that nanomaterial
research is similar to other chemical research, presumably indicating that they have other
chemical research practices under appropriate control. Answers 1 and 5 indicate they would
appreciate more specifically targeted work health and safety information.
2.4 Summary from the survey
a) There were 38 respondents to the survey, who reported working on a range of different
types of nanomaterials. The respondents organisations were primarily from universities,
commercial/industry and government research groups. The most common nanomaterials
handled are metal oxides, metals and carbon nanotubes and the most common areas of
application are into energy, medical, surface coating and textile uses.
b) Many organisations (27/35), and notably universities (20/21), manufacture their own
engineered nanomaterials. A significant number also purchase them from overseas or from
within Australia (see Figure 3).
c) A number of respondents obtained work health and safety information about the
nanomaterials that they are using from an MSDS. The main work health and safety issues
examined for engineered nanomaterials are handling and storage, physical and chemical
23
properties, toxicological data and exposure controls/PPE (see Table D). The available
information on these topics is limited.
d) Most respondents indicated that substitution/modification is used to change the
functional properties of the product (see Figure 4). A work sector analysis indicates that
substitution/modification occurs more in university research and less in commercial/industry
research which is as expected in product development.
e) The five properties that are manipulated by modifying or substituting engineered
nanomaterials by the highest number of organisations are particle size, physical properties,
agglomeration properties, chemical properties and conductive properties. A small number of
respondents indicated that they use substitution/modification to change the health hazard or
toxicological properties (see Figure 5).
f) Adding functional groups (17 responses) and modifying surface characteristics (16
responses) are the two most popular methods of substitution/modification of engineered
nanomaterials. Others include changing the form of the material, the particle size and shape
and the crystalline structure (see Figure 6).
g) Australias nanotechnology activities are generally at the early stage of nanomaterial
development, i.e. more focussed on de novo research than later stages of product
development/production. However substitution/modification methodologies are well known
and used in Australia and thus there is an existing capability that might be applied more
broadly to work health and safety related purposes.
24
25
Figure 7: Mechanisms of cell entry and uptake for engineered nanomaterials (Thurn
et al. 2007)
The evidence of these mechanisms for the uptake of engineered nanomaterials into cells
are summarised in Table F (Thurn et al. 2007). This information is key to understanding
why certain substitution/modification options are more effective than others and will be
highlighted throughout this review for specific processes.
In summary, the most critical characteristics that affect nanoparticle uptake by cells are the
particle size, surface charge or modification and the specific cell type involved. Studies
have indicated that changes in one (or more) of these parameters can cause major
differences in the efficiency and type of cellular uptake (Thurn et al. 2007).
26
Table F: Evidence for different cell uptake mechanisms derived from specific studies (adapted from Thurn et al. 2007)
Reference
Nanoparticle type
Cell type
Localisation
Uptake mechanism
Factors that
impact on
uptake
50 nm silica magnetic NP
24 and 43 nm polystyrene
100 nm PLGA
Endosomal
24 nm = perinuclear,
43 nm = lysosome
Membrane bound,
intracellular
RothenRutishauser et al.
(2006)
20 nm 1000 nm polystyrene
(+), (-) and uncharged; 25 nm
gold (+) and uncharged; 32 nm
TiO2
40 nm 4500nm polystyrene
particles
261 nm PVA-coated and 295
nm TPGS-coated PLGA
nanoparticles;
50 nm 1000 nm polystyrene
90-95 nm PLA/PEG-PLA
nanoparticles, (+) and (-)
charge
100 nm MSN uncoated;
with weak, moderate, and
strong (+) charge
78 nm 1000 nm polystyrene
Microsphere
Human RBC
Cytoplasm
CME
24 nm = CME independent,
43 nm = CME
Endocytosis of nanoparticles clathrin and caveolin
independent
Non-phagocytic uptake of NPs
200nm, irrespective of charge
NP size
NP size
Qaddoumi et al.
(2003)
Cytoplasm and
membrane bound
Cytoplasm and
nucleus
Not defined
NP size and
charge
NP size and
surface
modifications
Both perinuclear
Not defined
(+) NP = CME
(-) NP = Clathrin/caveolin
independent
hMSC: uncoated, weak, mod.
(+) = CME, strong (+)
unknown. 3T3-L1 = all CME
Macrophage:
1000nm = phagocytosis,
78 nm 200 nm = actinindependent;
RBC: all actin-independent
Foged et al.
(2005)
Win and Feng
(2005)
Harush-Frenkel et
al.
(2007)
Chung et
al.(2007)
Geiser et al.
(2005)
27
Intracellular, not
membrane-bound
Not defined
NP size
NP size
NP charge
NP charge and,
Cell-specific
effects
Cell-specific
effect
Reference
Nanoparticle type
Cell type
Localisation
Uptake mechanism
Factors that
impact on
uptake
Zheng et al.
(2005)
Steinhauser et al.
(2006)
de la Fuente and
Berry (2005)
22 nm folic acid-LDL NP
KB (FR+) human
epidermoid carcinoma cells
BT-474 and SK-BR-3
human breast cancer cells
hTERT-BJ1 human
fibroblast
Cytoplasm, not
nuclear
Not defined
Receptor-mediated endocytosis
Receptor mediated endocytosis
Nucleus
Not defined
Surface
modifications
Surface
modifications
Surface
modifications
220 nm Trastuzumab-HSA NP
2.8 nm HIV-Tat peptideconjugated gold NP
CME = clathrin-mediated endocytosis, FR+ = folate receptor positive, HIV-Tat = human immunodeficiency virus- transactivator of transcription,
hMSC = human mesenchymal stem cell, HSA = human serum albumin, LDL = low density lipoprotein, MSN = mesoporous silica nanoparticle, NP = nanoparticle,
PEG-PLA = poly(ethylene glycol-co-lactide), PLA = DL-polylactide, PLGA = poly(D,L-lactic-co-glycolic acid), PVA = polyvinyl alcohol, RBC = red blood cell,
TPGS = d-alpha-tocopheryl polyethylene glycol 1000 succinate, (+) = positively charged, (-) = negatively charged.
28
The individual endocytic pathways also have a defined specific size range of
engulfed particulate or soluble material (see Figure 8).
29
30
31
Surface charge also affects the interaction of nanomaterials with serum proteins
which may alter the nature of the particles surface that is presented to the cell
membrane prior to uptake, but can also ultimately influence their clearance from the
body. Recent studies involving intravenously-administered quantum dots (QDs) in
rodents have indicated some nanoparticle requirements for rapid renal filtration and
urinary excretion, i.e. Zwitterionic or neutral organic coatings of QDs prevented
adsorption of serum proteins that would otherwise increase the QD hydrodynamic
diameter by >15 nm and prevent renal excretion. Those QDs with a final
hydrodynamic diameter <5.5 nm exhibited rapid and efficient urinary excretion and
elimination from the body (Choi et al. 2007). Such findings show that the
hydrodynamic size of a positively charged nanoparticle within a biological system
may be much larger than the size of a pristine nanoparticle, due to proteinnanoparticle interactions. This would result in significantly slower renal clearance
resulting in blood half-life values of hours rather than minutes, with major implications
for the toxicokinetics of such nanoparticles that reach the circulatory system (Choi et
al. 2007).
3.1.3 Importance of cell specific effects for nanoparticle uptake
Overall, when employing the weight of evidence approach concerning cell-specific
effects of nanomaterials, a greater weight should be placed on data derived from in
vivo rather than in vitro studies. In regard to in vitro studies, the best evidence will
come from studies using human primary cells whereas a lower level of evidence is
provided by studies using immortalised/tumour cell lines and animal cells. The
premise is that the closer a study mimics a human system the more useful is the
data that it provides.
Different mechanisms and rates of uptake are exhibited by different cell types for the
same nanomaterial. For example, when Chung et al. (2007) exposed mouse
adipose/fibroblast 3T3-L1 and human mesenchymal stem cells (hMSC) to silica
mesoporous nanoparticles with strong positive surface charges, it was found that the
uptake mechanisms were cell type specific. The 3T3-L1 cells were found to utilise
clathrin-mediated endocytosis to take up the nanoparticles, whereas hMSC cells
used an undefined alternate mechanism. When the silica mesoporous nanoparticles
were uncoated, or had weak or moderate positive charges, the uptake for both celltypes was by clathrin-mediated endocytosis.
Geiser et al. (2005) performed an in vitro study of the uptake of fine (200 -1000 nm)
and ultra fine particles (UFP) (<100 nm) by human red blood cells (RBCs) and
porcine pulmonary macrophages. Using confocal laser scanning microscopy it was
found that 77% of macrophages contained UFPs and 56% contained fine particles.
However it was also found that only 40% of RBCs contained UFPs and none
contained no fine particles. This further demonstrates that particle uptake is cellspecific.
32
localisation within a targeted cell following the binding of the conjugated nanoparticle
to complementary DNA sequences at these sites (Paunesku et al. 2003, 2007; Qin
and Yung 2006).
3.1.5 Biocompatibility and surface coatings
When undertaking surface modification to change the efficiency and uptake of an
engineered nanomaterial there is also the consideration of biocompatibility, which is
important in negating or reducing potential toxic effects (Wang et al. 2004).
Biocompatibility has been defined as the property of being biologically compatible by
not producing a toxic, injurious, or immunological response in living tissue. A major
goal in substituting or modifying an engineered nanomaterial should be to make it
biocompatible. Therefore, any surface coating used with a nanomaterial should
confer this property.
An example of the application of the biocompatible principle is case of synthetic
parts (e.g. prostheses, artificial organs, contact lenses, artificial limbs, biosensors or
encapsulating membranes), which are now being used as replacements for
defective parts in human hosts (Mathieu 2001; Sabbatini and Zambonin 1996; Good
1993; Aleyamma and Sharma 1991; Makohliso 1999; Wang and Ruckenstein 1993).
The structural materials used in these replacement parts are usually in themselves
bio-incompatible. However, in order for them to be accepted in their host they need
to be made compatible. This has been done in many cases by grafting a
biocompatible surface material onto the replacement part, which has the effect of
preventing protein absorption and reduces or negates toxic effects of the bioincompatible material (Marieb 1998). Such biocompatible materials include
hydroxyapatite, chitosan, chitin coating, peptides, polysaccharides and other
polymeric materials.
There are a number of factors which determine the interaction of cells and
biomaterials. These include surface energy; the balance between surface
hydrophilicity and hydrophobicity; chemical structure and functional groups; type and
the density of surface charges; molecular weight and conformational flexibility of the
polymer; and surface topography and roughness (Wang et al. 2004). There has
been much time and effort invested in the health care industry to develop and
improve materials that are suitably biocompatible with the living environment
(Aleyamma and Sharma 1991). Synthetic polymers are a broad range of materials
that can easily be utilised as surface coatings. However, the issue of non-specific
protein adsorption needs to be addressed to prevent inflammation in order that these
materials can successfully become biocompatible. Therefore, research is directed
towards polymers which have minimal protein adsorption by using strategies that
mimic the external cell membrane or use biologically active materials such as
proteins, peptides or polysaccharides. This type of procedure has the advantage of
functionalising the surface of the material without modifying its bulk properties.
Immobilisation of biomolecules on the surface of materials may be achieved by
several mechanisms including covalent binding or physical adsorption (Mathieu
2001).
34
SWCNTs are composed of a single rolled up graphene sheet and have a diameter of
0.4 to 3 nm (see Figure 9). MWCNTs are composed of a concentric arrangement of
many cylinders and have a diameter up to 100 nm (see Figure 10). The structure of a
CNT can be specified based on the orientation of the tube axis with respect to the
hexagonal lattice through specification of its chiral vector using indices (n,m).
Classifications are described as armchair when n=m ((8,8) in Figure 9) and zigzag
35
when m=0 ((14,0) in Figure 9). There are three methods that have been established
for the production of SWCNTs and MWCNTs. See Table G for details of these.
Table G: Overview of the important synthesis procedures for CNTs
(Adapted from Table 1 of Balasubramanian and Burghard 2005)
Synthetic
method
Reaction principle
Average
diameter of the
tubes
Maximum
production
rate
Electric arc
discharge
1.31.4 nm
Laser ablation
1.4 nm
50 g per day
Catalytic
decomposition
of gaseous
hydrocarbons
Decomposition of a gaseous
hydrocarbon source (e.g. an
alkane or CO) is catalysed by
metal nanoparticles (Co or Fe).
Particles are prepared by
pyrolysis of suitable precursors
(e.g. [Fe(CO)5]) at 10001100C
under high pressure.
1 nm
50 kg per day
For each of the three production methods, there are issues with the purity of the CNTs, with
there being impurities such as metals (e.g. Fe or Co), fullerenes or amorphous carbon.
Consequently, purification of CNTs is required prior to further processing (Balasubramanian
and Burghard 2005) and typically involves the following steps:
a) thermal oxidation step in air
b) acid reflux step (using HCl)
c) filtration, and
d) thermal annealing.
The synthesis of large quantities of CNTs usually results in bundles of hundreds of CNTs
being produced. These bundles can easily be dispersed by ultrasonic treatment in aqueous
surfactant solution, which leads to individual CNTs being enclosed in a detergent shell
(Balasubramanian and Burghard 2005). An alternative method of preparing relatively pure
isolated CNTs is by vapour deposition onto a solid support (see Figure 11).
36
Figure 11: Chemical vapour deposition used for the synthesis of individual CNTs by
surface deposited catalyst particles (adapted from Balasubramanian and Burghard
2005)
CNTs have attracted much interest because their specific strength, lightness, electrical and
related properties may potentially be exploited in new products e.g. coloured textiles,
conductors, semiconductors, composites etc. Like fullerenes and quantum dots, these are a
new class of compound which are attractive because of their potential commercial
applications.
4.1.2 Toxicology of carbon nanotubes
There is now a growing body of information about the potential hazards of CNTs. Further
data is needed and it is noted that there are a number of difficulties associated with toxicity
testing for CNTs, which include:
a) fibre agglomeration and difficulty involved in isolating single fibres
b) ensuring CNTs come into contact with cells for in vitro tests
c) difficulties involved in using standard toxicity tests, e.g. inhalation exposure studies in rats
and mice failing because fibres did not get to the pleural cavity in the lungs, due to
difficulties in aerosolising the CNTs
d) problems of interpretation of test data, e.g. when in vivo intratracheal instillation is used,
excessive loads of CNTs can easily be deposited, which can lead to artificial biopersistence
in the lung and questionable results (Drew 2009).
Lam et al. (2006) in a review of the available literature on CNT toxicity concluded that the
results of rodent studies collectively showed that regardless of the process by which CNTs
were synthesised and the types of metal impurities that they contained, CNTs were capable
of producing inflammation, epithelioid granulomas, fibrosis and biochemical/toxicological
changes in the lung. They also indicated that SWCNTs were more toxic than quartz, a
known chronic workplace inhalation hazard, and carbon black which exhibited minimal lung
responses in comparative toxicological inhalation studies in mice. Lam et al. (2006) stated
that SWCNTs present in the lungs of mice produce respiratory impairment, damage DNA in
the aorta, increase aortic plaques, induce atherosclerotic lesions in arteries of the heart and
retard bacterial clearance.
There are two important in vivo studies that both studied the carcinogenic and fibrogenic
behaviour of MWCNTs in the intraperitoneal cavity of mice (Poland et al. 2008, Takagi et al.
2008). Whilst there were experimental difficulties with both studies, it was clearly shown by
Poland et al. (2008) that inflammatory and granuloma responses were associated with long
37
straight CNT fibres, having a structural aspect ratio similar to long fibre asbestos (Drew
2009). Curled-tangled CNT fibres did not cause the same effects and are considered to be
less pathogenic than long straight CNTs (Poland et al. 2008).
Donaldson et al. (2009) in a study of the hazards poised by high aspect ratio biopersisitent
nanoparticle fibres indicated that long fibre CNTs have a high potential to be retained in the
pleural membrane and to cause diseases such as mesothelioma. They made the following
conclusions:
a) CNTs have the length, thinness and biopersistence associated with pathogenic fibres.
b) CNTs can also be short and tangled in which case they do not pose a fibre hazard but
may pose a particle hazard.
c) A proportion of CNTs that are respirable and deposit in the distal airspaces are likely to
transit through the pleura.
d) Short CNT fibres (<5 m) will transit through the parietal pleural stomata. However, long
CNT fibres (> 15 m) can reach the pleura and be retained where they can cause
genotoxicity and inflammation.
e) Unmodified CNTs may be classified as durable and biopersisitent. However, if they are
defective, derivatised or otherwise weakened they may undergo some form of breakdown
and be less biopersisitent.
The potential mesothelioma hazard from CNTs was examined in detail by Drew (2009). For
MWCNTs, weight of evidence suggests that:
long thin MWCNTs (i.e. of pathogenic fibre dimensions) present a mesothelioma hazard
to workers if they are inhaled, and if sufficient numbers are in contact with mesothelial
tissue
MWCNTs that are not of pathogenic fibre dimensions do not have this hazard.
To date, there are no data on the potential of SWCNTs or functionalised CNTs to cause
pathogenic fibre-like responses, but there is no evidence that responses would be different
to MWCNTs.
Thus, as a precautionary default, Drew (2009) recommended that:
38
potentially lead to decreased toxicity by increasing the hydrophilic character and therefore
biocompatibility of the manufactured CNTs (Balasubramanian and Burghard 2005).
However, such modifications may impact on other properties, e.g. mechanical and electrical
properties (Chattopadhyay et al. 2008). Chemical modification is examined in detail in the
following sections.
4.1.3.1 Addition reactions of carbon nanotubes
Carbon nanotubes possess high chemical and mechanical stability, which is a useful
application property, but it does not allow for easy and controlled functional group addition
(Balasubramanian and Burghard 2005). However, there are several known reactions
presented in Table H below which give functional additions and increase the hydrophilic
character of the CNT moiety. The main concern is the low conversion/functionalisation rate
for these reactions due to the unreactive nature of CNTs. Other potential conversions are
presented in Figure 12. Overall, more addition reactions are known for SWCNTs than for
MWCNTs (Balasubramanian and Burghard 2005).
Figure 12: Potential addition reactions that can be used to functionalise nanotube
sidewalls (from Balasubramanian and Burghard 2005).
39
Table H: Known addition reactions for CNTs (summarised from Balasubramanian and Burghard 2005).
40
Type of
CNT
Modification
Product
Reagents/Process
SWCNTs
Carboxylation
Carboxyl SWCNT
(5-10%
conversion)
CarboxylSWCNTs
Anhydride
formation
Acid treatment
Nitrogen
doped
MWCNTs
SWCNTs
a) Carboxylation
b) Amidization
SWCNT
anhydride
(1-5%
conversion)
MWCNT ferritin
or BSA amide
Fluorination
Fluorinated
SWCNTs
(10-20%
conversion)
Conditions/Comments
a) End tubes open and holes form in
side walls followed by oxidative
etching of sidewalls
b) SWCNTs produced are100-300
nm in length with sidewalls
containing carboxyl groups
c) Further reactions of amide and
ester formation possible
d) Reaction occurs at wall defects
a) Ring closure reaction is possible
b) A mixture of products can be
obtained
c) Reaction occurs at wall defects
a) Reaction is a covalent binding of
protein to MWCNT
b) Reaction occurs at wall defects
a) SWCNTs used can be either from
laser ablation method or from the
high pressure carbon monoxide
(HIPCO) process
b) Helium gas is required for dilution
at elevated temperatures
c) Increased fluorination is observed
at higher temperatures
References
Zhang et al. 2003
Figure 13: Schematic view of carboxyl functionalised MWCNTs and the process of
their attachment to an aromatic polyamide (from Ge et al. 2005)
Ke et al. (2007) reported the covalent modification of MWCNTs with a low molecular
weight chitosan (LMCS) using a nucleophilic substitution reaction. A summary of the
synthetic method is provided by Figure 14 and images of the product in Figure 15. The
resulting grafted MWCNT was fully characterised by a series of methods including infrared
(IR), X-ray photo electron spectroscopy (XPS) and 13C nuclear magnetic resonance
(NMR). Ke et al. (2007) calculated that for every 1000 carbon atoms in the nanotube there
were be four molecular chains of chitosan (Ke et al. 2007).
Figure 14: Process of functionalisation and surface grafting of MWCNTs with low
molecular weight chitosan (from Ke et al. 2007)
41
Figure 15: TEM images of a) raw MWCNTs, b) cut and purified MWCNTs and c)
chitosan grafted MWCNTs. Arrows on c) indicate position of polymer
addition/modification (from Ke et al. 2007).
Dutta et al. (2007) investigated the toxicity of SWCNTs with and without a polymer coating.
The results indicated that precoating SWCNTs with a non-ionic surfactant (e.g. Pluronic
F127), inhibited albumin absorption and anti inflammatory response. Dutta et al. (2007)
suggested that the uptake of proteins (e.g. albumin) can be significantly reduced by a
coating present on the surface of CNTs.
Sayes et al. (2006a) demonstrated that the in vitro cytotoxic response of human dermal
fibroblast cells was dependent on the degree of functionalisation of SWCNTs. These
researchers showed that as the degree of sidewall functionalisation of SWCNTs increased
with the addition of phenyl-(COOH)2 or phenyl-SO3H moieties, the SWCNT sample
became less cytotoxic. This sidewall functionalisation was also substantially less cytotoxic
than surfactant-stabilised SWCNTs (Sayes et al. 2006a).
4.1.4 Impact of modification on potential exposure levels
Johnson et al. (2010) examined emission levels of various types of carbon nanoparticles in
laboratory activities. Both raw MWCNTs and MWCNTs modified with OH groups
(MWCNT-OH) were examined.
A. Weighing MWCNTs and transferring to mixing beaker inside a hood with the ventilation
switched off.
For particle size above 300nm (measured by hand held particle counter, HHPC),
emissions of MWCNT-OH were higher.
4.1.5 Conclusions
CNTs can be functionalised and surface-modified in order to increase their solubility and
biocompatibility. However, such modifications may impact on other properties (e.g.
mechanical properties). Further investigation of the toxicity of these modified CNTs needs
42
to be made to assess the extent of the reduction in potential workplace hazard. The size
control of CNTs is also important to control toxicity. Their chronic toxicity potential can be
reduced by using short CNTs and keeping their length to less than 5m.
However, these modifications may be incompatible with the intended use, for example, in
polymer or metal nanocomposites.
4.2 Fullerenes
4.2.1 Background
The carbon allotrope C60, usually referred to as a buckyball, was discovered in 1985 by
Smalley, Kroto and Curl from Sussex University and Rice University. A buckyball
resembles a European championship soccer ball in shape and was named
Buckminsterfullerene (see Figure 16). The molecule is geometrically shaped as a
truncated dodecahedron with a carbon atom sitting at each corner of the polyhedron
(Sayes et al. 2004). Fullerenes are currently being investigated for their potential medical
applications.
43
fullerenes were less efficient in producing reactive nitrogen and oxygen species
Sayes et al. (2004) showed that the in vitro cytotoxic concentration of fullerenes changed
over seven orders of magnitude with minor modifications of the fullerene that progressively
increased their solubility and reduced their toxicity. The C60 parent unsubstituted fullerene
exhibited the highest toxicity whereas more functionalised (and polar) derivatives showed
decreased toxicity, see Figure 17.
44
Figure 18: Some generalised reactions of C60 fullerenes (adapted from Taylor and
Walton (1993)).
45
46
d) Dermal absorption studies for nano TiO2 conclude consistently that TiO2 is not absorbed
through the skin.
e) Intravenous and oral toxicity studies have been used to investigate the systemic
distribution of TiO2. The oral studies indicated that nano TiO2 is absorbed from the
gastrointestinal tract and is distributed throughout the body. Intravenous and oral studies
resulted in TiO2 retention in the lung, kidney, liver and spleen but not in the lymph nodes,
brain, plasma or blood cells.
f) There is sufficient data to indicate that anatase nano TiO2 is more toxic/phototoxic than
the rutile nano material. However, both forms can induce a pulmonary inflammation
response if the concentration of exposure is high enough, with evidence for this being
provided by rat inhalation studies.
g) A variety of traditional hazard/safety tests using nano-TiO2 have shown the material to
be of low acute toxicity.
Relating to concerns about potential toxicity from the use of nano-TiO2 in sunscreens,
Barker and Branch (2008) reported on the increased incidence of unsightly appearance
defects of building materials due to nanoparticle-containing sunscreens. These defects
were on recently installed prepainted steel roofs and other durable surface coatings, and
were due to the aggressive activity of photocatalytic grades of nano TiO2 (especially
anatase) in sunscreens that had been transferred to the building materials during
construction handling. The photocatalytic particles had accelerated the weathering of the
steel surface by >100 fold. However, the relevance of these findings to human sunscreen
usage depends on long-term persistence of insoluble nano TiO2 on the skin following
sunscreen application, which is unlikely due to the constant shedding of dead skin cells.
4.3.3 Potential substitution/modification of nano titanium dioxide
Sayes et al. (2006b) characterised the in vitro cytotoxicity of nanoscale anatase and rutile
forms of TiO2 under ambient light conditions in cell culture. The authors found that
cytotoxicity and pro-inflammatory responses were observed for anatase only, and only at
relatively high concentrations (100 g/ml) and these were found to increase with dose and
the time of exposure. The phase composition of the crystalline structure, namely the
anatase or rutile form, was an important factor with anatase being up to 100 times more
cytotoxic than the rutile form (see Table I). The most cytotoxic form of nano TiO2 was
found to be the most efficient at generating reactive oxygen species (ROS) in cell cultures.
These results are important because they indicate that different crystalline forms of the
same nanomaterial can exhibit dissimilar toxicological properties (Sayes et al. 2006b).
47
Table I: Summary of results for correlating nanoscale TiO2 structure with toxicity in
cells (Adapted from Table 3 of Sayes et al. 2006b)
Particle
sample
Photoactive
Nano-TiO2
Anatase
particles
Yes.
Yes
Up to 100 times
greater than
rutile.
Generates
ROS
No
Produces
cytotoxic
response
(LC50)
1500 g/ml
Induces
membrane
leakage
Decreases
mitochondrial
activity
Produces
pro-inflammatory
response
1500 g/ml
(w/o light)
30 g/ml
(w/light)
1500 g/ml
300 g/ml
N/A
N/A
N/A
N/A
N/A= not applicable - does not generate ROS (reactive oxygen species)
w/o= without
w/ = with
The results of Sayes et al. (2006b) experimentally validated other findings for the nano
TiO2 anatase and rutile phases. Selloni et al. (1998) and Vittadini et al. (1998) both
showed that anatase and rutile particles differ significantly in their surface chemistry,
specifically in relation to generating ROS. First principle calculations by these authors
indicated that water molecules dissociatively absorb onto anatase surfaces, but not
onto rutile surfaces. When water absorbed on to an anatase surface is illuminated with
UV light, reactive photocarriers are trapped on the surface and come into contact with
Ti-OH species, which then react to form hydroxyl radicals that are the primary oxidising
species in oxidative photocatalytic processes. These findings are supported by the
study of Jiang et al. (2008a), which demonstrated the oxidant generating capacity for
different crystal phases of TiO2 nanoparticles was highest for amorphous, followed by
anatase, and then anatase/rutile mixtures, and lowest for rutile samples.
The paint industry has developed many methods for coating macroscopic TiO2 in order
to decrease its photoactivity. However, these are generally not compatible for use with
nanoparticulate TiO2. At this stage there are relatively few methods to introduce robust
surface coatings onto nano TiO2.
4.3.4 Conclusions
It can be concluded that when formulating a new nano TiO2 product or use, its potential
toxicity can be controlled by varying the crystalline form used. For example, the UVphotocatalytic activity of nano TiO2 can be reduced by partial or full replacement of the
anatase form with the less reactive rutile form, especially in sunscreen applications and
personal care products. In applications involving the use of an industrial-grade
photocatalyst, such as the coatings on self-cleaning glass surfaces, then the potential for
shedding of anatase nanoparticles into the environment needs to be assessed and
minimised to reduce environmental impact.
48
49
Das et al. (2007) further demonstrated that nano ceria particles (2-5 nm) exhibited
autocatalytic properties which increased the survival of cultured adult rat spinal cord
neurons in an in vitro model. The neurons that had been treated with nano ceria
demonstrated normal electrical functioning similar to untreated neurons. This is indicative
of functional biocompatibility (see Figure 19). Cells treated with nano ceria also showed
higher cell survival rates in the adult rat spinal cord model system with or without hydrogen
peroxide-induced oxidative injury. From this research Das et al. (2007) concluded that
nano ceria autocatalytic particles could be used for the in vivo repair of spinal cord
neurons and for other in vivo applications such as the use of nano ceria for drug delivery
and imaging applications. Das et al. (2007) suggested a detailed pathway for the
regenerative properties of nano ceria and a probable mechanism for its free radical
scavenging property and auto-catalytic behaviour (see Figure 19).
50
4.5.1 Background
Zinc oxide (ZnO) is a semi conductor and is an important material that is used in
applications such as cosmetic materials, catalysts, luminescent and piezoelectric devices,
photovoltaic solar cells, chemical sensors and varistors (Grasset et al. 2003). Research on
thin film zinc oxide materials has significantly increased over the last 10 years after it was
found that high quality epitaxial ZnO thin films display excitronic UV laser action at room
temperature which indicates the presence of closely packed hexagonally shaped micro
crystallites in these films and potential use as a semi-conductor. ZnO together with TiO2
51
have a band gap energy close to 3 eV, i.e. their absorption of UV light almost extends up
into the visible region, and a high refractive index. Thus, they are the normal substances
used in broad spectrum sunscreens; an application of particular importance in the
Australian environment (Grasset et al. 2003).
4.5.2 Toxicology of nano zinc oxide
In a general review of the toxicology of engineered nanomaterials, Drew (2009) reported a
number of findings from research involving ZnO nanoparticles and micron-sized particles:
the weight of evidence indicated that insoluble nano ZnO when applied to the skin
remains on the surface and in the outer layer, and does not penetrate through the
stratum cornea
a European evaluation which reported that micron-sized ZnO has been found to be
clastogenic, possibly aneugenic, and inducing DNA damage in cultured mammalian
cells in vitro under the influence of UV light
no evidence was found of phototoxicity on intact skin from studies involving human
volunteers
Furthermore, there are issues with the high concentrations used in many in vitro
studies that report the cytotoxicity of nano ZnO. In addition, there is the possibility that
the minor increases in genotoxic potency from co-exposure to ZnO and UV irradiation
may not necessarily represent a true photo-genotoxic effect, but may occur due to an
increased sensitivity of the test system subsequent to UV irradiation (Dufour et al.
2006).
Interestingly, the recent report by Barker and Branch (2008) about the photocatalytic
activity of metal oxide nanoparticles in some sunscreens also included a sample
containing ZnO. However, the relevance of these findings to human sunscreen usage
is highly unlikely, as ZnO is much more water soluble than TiO2 and far less likely to
persist on the skin as intact nanoparticles following sunscreen application.
While the review of Drew (2009) and other research suggest there is low risk from the use
of nano ZnO in sunscreens or cosmetic applications, there are ways to reduce the toxicity
of the material as described in the following sections.
4.5.3 Potential substitution/modification of nano zinc oxide
Grasset et al. (2003) used aminopropyltriethoxysilane (APTES) to coat 20-30 nm ZnO
nano particles. These researchers used three processes (basic, acidic or toluene based)
to perform the surface modification, and the resultant samples were characterised for their
optical, structural and grafting (coating) properties using scanning electron microscopy
(SEM), TEM and X-ray diffraction. Using diffuse reflectance measurements it was noted
that the silane coating did not modify the transmittance spectra of ZnO (see Figure 21). It
was suggested that the coated form of nano ZnO should be used in preference to
uncoated nano ZnO particles, due to its enhanced grafting, structural and optical
properties (Grasset et al. 2003).
52
Figure 21: Optical transmittance of the non-grafted and the grafted zinc oxide
powder (taken from Grasset et al. 2003)
Hong et al. (2006, 2009) reported that nano ZnO particles prepared by direct precipitation
from ammonium carbonate and zinc carbonate and then calcinated at 450C for 3 hours
can be surface modified by capping with oleic acid, coating with SiO2 or grafting with
polystyrene. It can be seen from UV-visible absorption spectra of methyl orange (see
Figure 22) that surface catalytic degradation decreases when nano ZnO is surface
modified with SiO2. This is indicative of lower cytotoxicity for the surface modified nano
ZnO.
Figure 22: UVvisible light absorption of methyl orange solution vs. time using ZnO
and ZnO/SiO2 (from Hong et al. 2006).
Wu et al. (2007, 2008) synthesised biocompatible nano ZnO particles (2-5nm) doped with
Co, Cu and Ni cations and surface-capped with two types of aminosilanes and titania.
53
Synthesis was by a colloidal soft chemical process. The electrical, optical and magnetic
properties of ZnO can be adjusted by doping of the ZnO lattice structure. Dual colour
bioimaging on human cells (U-937 histiocytic lymphoma and MG-63 osteosarcoma cells)
showed turquoise emission at the cytoplasm and blue emission at the nucleus
simultaneously, with high photoluminescence emissions at the blue-violet and UV
wavelength ranges. Mung bean seedlings labelled by synthesised ZnO nanocrystals gave
a bright green emission. Cytotoxicity tests showed that the aminosilane-capped
nanoparticles have little toxicity or are non-toxic. Quantum yields of 80-95% were typical
for these nanocrystals. These results show the potential for live imaging of both plant
systems and human cells using ZnO and Co-doped ZnO nanocrystals.
4.5.4 Conclusions
It can be concluded from the review of Drew (2009) that ZnO used in sunscreen type
products and for other similar applications exhibits a low level of toxicity and penetration in
to the human body. From the work of Grasset et al. (2003) and Hong et al. (2006, 2009),
there are potential surface modification options available for ZnO which have the potential
to reduce toxicity further. The work of Wu et al. (2007, 2008) indicates the potential live
cell imaging opportunities for ZnO particles with a doped crystalline lattice, that have also
been made more biocompatible by surface modification.
4.6 Nano gold (Au)
4.6.1 Background
Gold (Au) nanoparticles are used for a variety of applications including bioimaging,
biosensing and drug delivery (West et al. 2003; Mann et al. 2000 and Hermanson 1996).
Hybridisation assays, flow cytometry and immunobloting are three of the applications that
rely on the excellent detection capabilities derived from nano Aus optical and contrast
properties (Demers et al. 2000; Thanh and Rosenzweig 2002). Nano Au is also receiving
attention for potential medical radiation applications e.g. as a contrast agent for tumour
detection and destruction, due to its radio-thermal properties. Two major issues involved in
using nano Au are insuring biocompatibility and aqueous solubility because biological
processes usually occur on a hydrophilic surface in an aqueous environment. Colloidal Au
is a commercially available product but is unstable and aggregates when electrolytic
solutions are introduced. In order to stabilise nano Au it may be coated with surfactant
molecules (Eychmuller and Rogach 2000; Tan and Zhang 2005).
4.6.2 Toxicology of nano gold
Nano Au has been used for nuclear transfection and targeting in non-viral gene
delivery applications because they easily enter cells, but their in vitro cytotoxicity in
certain cell types at high concentrations has limited the use of uncoated nano Au and
prompted the search for more biocompatible coatings that also aid in cell uptake
(Lewinski et al. 2008). There is also evidence of other adverse effects, such as the
potential for affecting immunological responses (Shukla et al. 2005). Studies of the
cytotoxic potential of nano Au particles, shells and rods have been reviewed by
Lewinski et al. (2008).
54
Li et al. (2008) assessed the potential cytotoxicity of nano Au using MRC-5 human fetal
lung fibroblast cells which were exposed to several different concentrations of 20 nm nano
Au particles. The researchers found that whilst nano Au had been taken up by the
fibroblasts, there were no visible alterations in cell morphology between the control and
the treated groups when examined by TEM. Nano Au taken up by the fibroblasts could be
easily identified forming clusters in a dose-dependent manner inside cellular vesicles (see
Figure 23). In order to examine nano Au induced oxidative damage, Li et al. (2008)
analysed the quantity of 8 hydroxydeoxyguanosine which is a known measure of oxidative
stress in cells. Nano Au induced oxidative damage at the highest concentration of 1 nM,
with a 5-fold increase in 8 hydroxydeoxyguanosine levels over control cells, while the
medium concentration of 0.5 nM did not alter this parameter. Li et al. (2008) concluded
that nano Au induces cytotoxicity in human lung fibroblasts by causing oxidative damage.
The authors also showed that nano Au inhibited cell proliferation and down-regulated cell
cycle genes whilst also affecting genes associated with DNA repair and genomic stability.
.
Figure 23: TEM image of MRC-5 human lung fibroblasts after 72 h culture with 1 nM
nano gold; arrows indicate the presence of Au nanoparticles in vesicles which
cluster around the nucleus (from Li et al. 2008).
As with many metal and metal oxide nanomaterials, differences in particle size and
surface modification of gold nanoparticles can alter their internalisation after binding to
cells, and the subsequent in vitro cytotoxicity (Jiang et al. 2008b). For example, Uboldi et
al. (2008) found that surface modification of nano gold (5-25 nm) with sodium citrate
impaired cell viability and proliferation greater than unmodified nanoparticles, in A549 and
NCIH441 human alveolar type-II cell lines exposed in vitro.
A previous review by Murphy et al. (2008) summarised several studies of the toxicity of
surface-modified nano Au particles; a summary is provided in Table J. Toxicity of surface
modified nano Au depends on particle size, shape, surface group types and cell type.
Two of these research groups reported that gold nanorods capped with
cetyltrimethylammonium bromide (CTAB) were more cytotoxic than overcoating nanorods
with polyethylene glycol (PEG) (Niidome et al. 2006) or phosphatidylcholine (Takahashi et
al. 2006). The process of PEGylation is known to decrease the non-specific binding of
55
molecules to a surface and in the case of gold nanoparticles, it reduces cell uptake to only
6% compared to uncoated particles. The effect of CTAB treatment of nanoparticles may
be due to unbound molecules in the sample and its lower order of biocompatibility
compared to the other capping agents, as CTAB is also a detergent used as a bactericide.
Table J: Summary of selected cytotoxicity data for surface modified gold
nanoparticles, 2004-2007 (adapted from Murphy et al. 2008)
Source
Size (nm)
Shape
Surface group
modification
Cell line
Goodman et al
(2004)
sphere
quaternary
ammonium (cation),
carboxylate (anion)
Connor et al.
(2005)
4, 12, 18
sphere
Cationic nanoparticles
were >7 fold more toxic
than anionic particles of
the same size.
None of these spherical
nanoparticles were toxic
at the micromolar ranges
used.
Shukla et al.
(2005)
3.5 0.7
sphere
citrate, cysteine,
glucose, biotin,
cetyltrimethyl
ammonium bromide
(CTAB)
lysine,
poly(L-lysine)
RAW264.7 mouse
macrophage cells
Niidome et al.
(2006)
65 5 x
11 1
rod
CTAB, polyethylene
glycol (PEG)
Takahashi et al 65 11
(2006)
rod
phosphatidylcholine
HeLa cells
Huff et al.
(2007)
50 long
rod
CTAB, PEG
KB human oral
epithelial tumor cells
Khan et al.
(2007)
18
sphere
citrate
HeLa cells
Patra et al.
(2007)
33
sphere
CTAB, citrate
56
Toxicity potential
57
4.6.4 Conclusions
It is possible to use surface coatings (e.g. phosphatidylcholine) or encapsulation with
biocompatible polymers (e.g. chitosan or polyethylene glycol) to reduce the toxic
potential of nano gold, whilst retaining its functionality and useability. Alkanethiolcapping may be used to increase biocompatibility and also functionalise the nano gold
for a range of biomedical applications.
58
al. 2005). Nano Ag coated with silica, functionalised with aldehyde groups and bioconjugated with oligonucleotides, has been used to colorimetrically detect DNA (Liu et al.
2005). Previously referred to as colloidal silver, nano Ag possesses antimicrobial activity,
and has been used for a long time to reduce the number and severity of infections in the
treatment of burns, sterilise surfaces such as medical implements (e.g. catheters), and
more recently to control bacterial growth on appliance and tool surfaces in the food
industry (Ulkur et al. 2005; Samuel and Guggenbichler 2004; Ahamed et al. 2008).
4.7.2 Toxicology of nano silver
Wijnhoven et al. (2009) note that one of the main functions of nano Ag is its antimicrobial
toxicity and therefore it should be regarded as a toxic agent. Wijnhoven et al. (2009)
reviewed the available data on the toxicity of nano Ag, and indicated that long term high
dose studies using particles of different sizes are required to ascertain its degree of
toxicity, because its antimicrobial activity is due to the release of Ag+ ions, which is
dependent on both the size and dispersal of nano Ag.
The subchronic inhalation toxicity of nano Ag (18-19 nm) was studied in Sprague-Dawley
rats by Sung et al. (2009). The animals were exposed to nano Ag (average diameter 18
19 nm) for six hours/day, five days/week, for 13 weeks in a whole-body inhalation
chamber. A dose-dependent increase in bile-duct hyperplasia in the liver was seen in both
the male and female rats, while mixed inflammatory cell infiltrate, chronic alveolar
inflammation, and small granulomatous lesions were seen in the lungs. Target organs for
silver nanoparticles were considered to be the lungs and liver in the male and female rats.
A no observable adverse effect level (NOAEL) of 100 g/m3 is suggested from the
experiments.
The oral toxicity of nano Ag (60 nm) was studied by Kim et al. (2008) over a period of 28
days (at 30, 300 and 1000 mg/kg/day) in Sprague-Dawley rats following the Organization
for Economic Cooperation and Development (OECD) Test Guideline 407. The results
indicated that nano Ag do not induce genetic toxicity in male and female rat bone marrow
in vivo, but nonetheless, the tissue distribution of nano Ag did show a dose-dependent
accumulation of silver content in all the tissues examined. In particular, a gender-related
difference in the accumulation of silver was noted in the kidneys, with a two-fold increase
in the female kidneys when compared with the male kidneys.
4.7.3 Potential substitution/modification of nano silver
Chung et al. (2008) surface modified nano Ag using phospholipids containing disulfide
groups. Biocompatible nanoclusters were formed when sodium borohydride was used to
reduce both silver ions and sulfide bonds (see Figure 26). TEM and optical absorption
spectra determined that well-dispersed nanoclusters of 3.8 nm diameter were formed with
a phospholipid/Ag ratio of 0.4. The elemental and molecular structure of the nanoparticles
were characterised by XPS and Fourier Transform Infrared (FTIR) with biocompatibility
being assessed using cell culture tests. Nanoclusters became internalised into fibroblast
or platelet cells after a short period of incubation and cells remained unharmed.
As with nano gold, alkanethiol-capping may also be used to increase the
biocompatibility of nano Ag, but such surface modifications would also decrease its
antibacterial activity, and potentially reduce the bactericidal usefulness of the material.
59
Figure 26: Illustration for the three phospholipids derivatives DSPC, DSPE, DSPBr
and their one-step reduction for manufacturing water-soluble silver nanoparticles
(adapted from Chung et al. 2008).
4.7.4 Conclusions
Nano Ag can be surface modified with hydrophilic groups, such as phosphorylcholine or
phosphorylethanolamine, to increase biocompatibility. Such modifications would also
decrease its antibacterial activity and potential usefulness in many current applications.
However, further functionalisation of biocompatible forms of nano Ag may provide potential
new applications, such as in biomedical diagnostics and biosensors.
60
the passive and unreactive nature of nano silica in redox and acid-base reactions
together with low surface charge density (pH<8)
61
small particle size (average ~9.1 nm) which form aggregates through hydrogen and
siloxane bonds
nano silica interacts with certain membrane structures of micro-organisms and cells
(e.g. integrin proteins)
nano silica particles are easily transported in aqueous media because of fast
diffusion
substances are easily adsorbed onto the external surface of nonporous primary nano
silica particles
there are changes in the interfacial water structure several nanometres thick above
the nano silica surface.
The authors noted that the surface modification of nano silica with proteins or polymers
(such as polyvinylpyrrolidone, PVP and PEG) leads to destruction of the secondary
structures and the formation of new secondary structures (50-100 nm). These new
structures reduced the diffusion of modified nano silica particles due to the formation of an
outer adsorption layer which leads to greater biocompatibility (Gunko et al. 2006). The
authors gave the example of the haemolysis of red blood cells being reduced if the nano
silica used is surface modified with proteins or polymers such as PVA (see Figure 27).
Such partial hydrophobisation by polymer coating or alkylsilylation causes reduced
hydrophilicity of the nano silica, which increases particle aggregation and reduces direct
membrane effects (Gunko et al. 2006).
Figure 27: Influence of PVA immobilised onto nanosilica A-300 on red blood cells
hemolysis as a function of PVA amount (last point corresponds to pure PVA
solution without silica) (taken from Gunko et al. 2006).
4.8.4 Conclusions
It is possible to modify the surface of nano silica with alkylsilylation, polymers or proteins to
increase its hydrophobic character, causing increased particle aggregation and reduced
direct membrane effects, and thereby improving its biocompatibility. Due to potential
toxicity of silica nanomaterials with high aspect ratios, consideration should also be made
as to whether nanowires may be substituted with nanospheres, while retaining
functionality for a particular application.
62
4.9.1 Background
Quantum dots (QDs) are semi-conducting nano crystalline structures, ranging from 2 to
100 nm depending on the types of surface coating or functional group added (Lewinski et
al. 2008), made from materials such as cadmium selenide or cadmium telluride (Tan and
Zhang 2005). For biological applications, QDs typically have a core/shell conjugate
structure, with the core composed of atoms from groups IIVI (e.g. CdSe, CdTe, CdS,
PbSe, ZnS, and ZnSe) and groups IIIV (e.g. GaAs, GaN, InP, and InAs) on the periodic
table (Lewinski et al. 2008).
QDs absorb white light which is then re-emitted as a specific colour after a couple of
nanoseconds. QDs range from 10 to 250 atoms in diameter and contain anywhere from
100 to 20000 electrons. Energy levels of QDs can be controlled by changing their shape
and size in order to control the depth of the potential. The science of QDs started to
develop during the 1980s when their potential was realised to build nano-scale computer
applications in which light is used to process information. Since this time more recent
medical applications have included molecular tagging in order that proteins, viruses,
antibodies or DNA can be tracked in the human body. In these applications, QDs behave
as a fluorophore absorbing energy at a particular wavelength and re-emitting energy at a
different longer wavelength (Tan and Zhang 2005).
4.9.2 Toxicology of quantum dots
Many of the core elements in QDs are known to be toxic at low concentrations, e.g.
cadmium, selenium, lead and arsenic. Therefore, stability is a critical factor in the cytotoxic
potential of QDs, as toxicity will eventuate from the release of toxic metal ions under
conditions that promote QD degradation, such as an oxidative environment (Lewinski et al.
2008).
Bare QD cores, and some QDs with biodegradable shells, are readily endocytosed by
cells into endosomes, which are then trafficked to various cellular compartments.
These include the acidic (~pH5) and oxidative environments of lysosomes and
peroxisomes, which can degrade QDs and result in the leaching of toxic metals from
the core (Chang et al. 2006). Studies of the cytotoxic potential of QDs have mainly
concerned CdSe/ZnS cores (recently reviewed by Lewinski et al. 2008), as these are
considered the most versatile for biological applications.
In a general review of the toxicology of QDs by Drew (2009), a number of conclusions
were made including:
the metal core of QDs can easily be exposed and can be a means by which toxicity
can occur if the body is exposed to this material
internalisation of uncoated QDs led to leaching of both cadmium and selenium from
the QD core in the low pH of endosomes
QDs are recognised in many instances as being foreign to the body and may
therefore be sequestered by the reticuloendothelial systems in the major organs
63
QDs do not appear to cause acute toxicity after being injected into the venous
system, however this does depend on whether QDs are internalised by cells
the core metal cadmium along with other similar heavy metals are known toxic
agents in most biological systems.
64
65
66
Suggested Strategies
Carbonaceous NPs
Carbon
Increase hydrophilicity by surface
modification in order to decrease
nanotubes
toxicity and increase biocompatibility.
Modify biodistribution to reduce
persistence.
Keep CNTs shorter than 5 m in
length.
Fullerenes
Substitution/Modification
Options
References
Other NPs
Silica
67
Partial hydrophobisation by
polymer coating or silylation.
Quantum
dots
68
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3. If you have extra information about the type(s) of materials you work with, please
write it here. For example, if you work with metal oxides, please describe the type of
oxide, e.g. zinc oxide or titanium dioxide.
5. How do you obtain the engineered nanomaterials? Please tick all options that apply.
Do you..
Obtain them from within Australia
Obtain them from overseas
Manufacture them
6. If you purchase engineered nanomaterials, what health and safety issues do you
consider when deciding which materials to purchase?
7. If you manufacture the nanomaterials, what health and safety issues do you consider
in their design?
8. If you obtain the nanomaterials from Australia or overseas, do you use modification
and/or substitution to change their attributes/properties?
Yes
No (If answer is no, please go to question 11)
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12. Would you be willing for us to contact you directly to discuss your work involving
engineered nanomaterials?
If yes, please provide us with your contact details:
Name:
Email Address:
Phone Number:
Done
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